from the edge of swimming/swarming halos using agar concentrations ranging from 0.4% to 0.7% by light and electron microscopy. Figure 2 shows that at agar concentrations of 0.4%, V. shilonii cells show a single-sheathed polar flagellum that is also observed in liquid cultures (See Erlotinib molecular weight Fig. 1a). Thinner structures compatible in diameter (c. 15 nm) with lateral flagella become observable if the cells are seeded in agar concentrations of 0.5% or 0.6%; however, under these conditions, the polar flagellum is still present (Fig. 2). At these agar concentrations, cells elongate, reaching an average size of 5 μm, although larger cells could be observed (data not shown). A notable reduction in the swarm diameter was observed Romidepsin at 0.7% agar; the cells obtained from this condition lost their flagella and most of them became round (Fig. 2). In order to determine the viability of V. shilonii cells after incubation in 0.7% swarming plates, we plated cells obtained from this condition on a solid medium and also inoculated them in a liquid growth medium.
Incubation was carried out overnight at 30 °C. Under both the conditions, the cells showed normal growth rates (data not shown). In general, Vibrio use the sheathed polar flagellum to swim. Rotation of this flagellum is powered by a sodium electrochemical gradient as shown by its sensitivity to amiloride (Fig. 1b). Given that at 0.5% agar both polar and lateral flagella are present (see Fig. 2), we tested whether the polar flagellum contributes towards expanding the swarm ring at 0.5% agar. The sodium channel blocker amiloride was added to 0.5% soft agar plates to inhibit the Na-dependent rotation of the polar flagellum. Figure 3 shows a slight reduction in the diameter of the swarm ring in the presence of 2 mM amiloride. This slight reduction in swarm diameter is statistically significant when compared with the control conditions either in the absence click here or in the presence of 2% DMSO. These findings suggest that the contribution of the polar flagellum to swarming in 0.5% agar is marginal and that this behavior is mainly dependent on the lateral flagellum
that seems to be insensitive to Na blockers. We isolated the flagellar basal-body complex following the procedure detailed in Materials and methods. The integrity of the isolated complexes was confirmed by electron microscopy. Figure 4a (left panel) shows the HBB structures stained with 2% ammonium hepta-molibdate (pH 8.0). Using this staining method, the flagellar filaments are preserved and very long filaments can be observed. In contrast, when filament–HBB samples were stained using 1% uranyl acetate, the flagellar filaments were lost, whereas the rest of the structure was preserved (Fig. 4a right panel). Filament–HBB samples were run in SDS-PAGE gels and the apparent molecular masses of the components were calculated (Fig. 4b).